Specific Increase in MDR1 Mediated Drug-Efflux in Human Brain Endothelial Cells following Co-Exposure to HIV-1 and Saquinavir

Persistence of HIV-1 reservoirs within the Central Nervous System (CNS) remains a significant challenge to the efficacy of potent anti-HIV-1 drugs. The primary human Brain Microvascular Endothelial Cells (HBMVEC) constitutes the Blood Brain Barrier (BBB) which interferes with anti-HIV drug delivery into the CNS. The ATP binding cassette (ABC) transporters expressed on HBMVEC can efflux HIV-1 protease inhibitors (HPI), enabling the persistence of HIV-1 in CNS. Constitutive low level expression of several ABC-transporters, such as MDR1 (a.k.a. P-gp) and MRPs are documented in HBMVEC. Although it is recognized that inflammatory cytokines and exposure to xenobiotic drug substrates (e.g HPI) can augment the expression of these transporters, it is not known whether concomitant exposure to virus and anti-retroviral drugs can increase drug-efflux functions in HBMVEC. Our in vitro studies showed that exposure of HBMVEC to HIV-1 significantly up-regulates both MDR1 gene expression and protein levels; however, no significant increases in either MRP-1 or MRP-2 were observed. Furthermore, calcein-AM dye-efflux assays using HBMVEC showed that, compared to virus exposure alone, the MDR1 mediated drug-efflux function was significantly induced following concomitant exposure to both HIV-1 and saquinavir (SQV). This increase in MDR1 mediated drug-efflux was further substantiated via increased intracellular retention of radiolabeled [3H-] SQV. The crucial role of MDR1 in 3H-SQV efflux from HBMVEC was further confirmed by using both a MDR1 specific blocker (PSC-833) and MDR1 specific siRNAs. Therefore, MDR1 specific drug-efflux function increases in HBMVEC following co-exposure to HIV-1 and SQV which can reduce the penetration of HPIs into the infected brain reservoirs of HIV-1. A targeted suppression of MDR1 in the BBB may thus provide a novel strategy to suppress residual viral replication in the CNS, by augmenting the therapeutic efficacy of HAART drugs.

Linker-scanning analysis of the HIV-1: integrase protein

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.

Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1-transmitting mothers and 165 propensity score-matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1-infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3-specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.

Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1.

UNLABELLED: Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response. IMPORTANCE: Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.

Polyploidy and Mitotic Cell Death are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells

Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy.

Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.

Shared monocyte subset phenotypes in HIV-1 infection and in uninfected subjects with acute coronary syndrome.

The mechanisms responsible for increased cardiovascular risk associated with HIV-1 infection are incompletely defined. Using flow cytometry, in the present study, we examined activation phenotypes of monocyte subpopulations in patients with HIV-1 infection or acute coronary syndrome to find common cellular profiles. Nonclassic (CD14(+)CD16(++)) and intermediate (CD14(++)CD16(+)) monocytes are proportionally increased and express high levels of tissue factor and CD62P in HIV-1 infection. These proportions are related to viremia, T-cell activation, and plasma levels of IL-6. In vitro exposure of whole blood samples from uninfected control donors to lipopolysaccharide increased surface tissue factor expression on all monocyte subsets, but exposure to HIV-1 resulted in activation only of nonclassic monocytes. Remarkably, the profile of monocyte activation in uncontrolled HIV-1 disease mirrors that of acute coronary syndrome in uninfected persons. Therefore, drivers of immune activation and inflammation in HIV-1 disease may alter monocyte subpopulations and activation phenotype, contributing to a pro-atherothrombotic state that may drive cardiovascular risk in HIV-1 infection.

Linker-scanning analysis of the HIV-1: integrase protein

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.

Integrase mutants defective for interaction with LEDGF/p75 are impaired in chromosome tethering and HIV-1 replication

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.

Nuclear import of HIV-1 integrase is inhibited in vitro by styrylquinoline derivatives

Nuclear import of HIV-1 preintegration complexes (PICs) allows the virus to infect nondividing cells. Integrase (IN), the PIC-associated viral enzyme responsible for the integration of the viral genome into the host cell DNA, displays karyophilic properties and has been proposed to participate to the nuclear import of the PIC. Styrylquinolines (SQs) have been shown to block viral replication at nontoxic concentrations and to inhibit IN 3'-processing activity in vitro by competing with the DNA substrate binding. However, several lines of evidence suggested that SQs could have a postentry, preintegrative antiviral effect in infected cells. To gain new insights on the mechanism of their antiviral activity, SQs were assayed for their ability to affect nuclear import of HIV-1 IN and compared with the effect of a specific strand transfer inhibitor. Using an in vitro transport assay, we have previously shown that IN import is a saturable mechanism, thus showing that a limiting cellular factor is involved in this process. We now demonstrate that SQs specifically and efficiently inhibit in vitro nuclear import of IN without affecting other import pathways, whereas a specific strand transfer inhibitor does not affect IN import. These data suggest that SQs not only inhibit IN-DNA interaction but would also inhibit the interaction between IN and the cellular factor required for its nuclear import.

Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Characterization of the nuclear import pathway for HIV-1 integrase

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.